(a) Field of the Invention
The present invention relates to a method of controlling the growth, size, and distribution of a lipid domain in a lipid layer using a substrate on which a topographic structure is formed, and a method of preparing a membrane device including a lipid layer having a lipid domain, where the growth, size, and distribution of the lipid domain can be controlled by said method, and a membrane device prepared thereby.
(b) Description of the Related Art
Recently, researches in the field of biotechnology in the post genome era have been concentrated on the study of proteins, which are known to regulate most of the biological processes in cells, in order to delineate the function of genes that encode the proteins and have been discovered through various studies including the Human Genome Project. However, the protein studies are partly hampered by the fact that the deduction of protein functions directly from the nucleotide sequences encoding them is limited, and more importantly, proteins need to adopt a correct tertiary and/or quaternary conformation for reliable studies, which is known to be difficult to provide in vitro. It is the tertiary structure of proteins, which mature proteins should adopt in cells through a process called “protein-folding” under various physiological conditions, not the primary structure of proteins, i.e., amino acid sequences, that is critical for proper functional studies of proteins. The importance of the tertiary structures are manifested by the development of a disease caused by a malfunctioned protein due to its abnormal tertiary conformation, which is usually caused by a genetic defect or other external factors.
Proteins implicated in the development of diseases have become an important target for the development of novel therapeutic agents, as well as being the basis for studies to understand disease-causing mechanisms.
The majority of proteins conventionally studied in relation to the development of a disease belong to a class of protein called a water-soluble protein. Among them are proteases, phosphatases, and kinases that are known to regulate the function of proteins by modulating a degradation, synthesis, and/or phosphorylation thereof. However, there are many other proteins that are also known to be involved in causing a disease and belong to another class of protein called a membrane protein, which is located in cell membranes and the study of which is less progressed than the study of water-soluble proteins. This is partly because membrane proteins are not amenable to isolation in a pure form, which is often a prerequisite for the study thereof. This is in contrast to water-soluble proteins, which are relatively easy to synthesize and purify in large quantities from prokaryotic and eukaryotic cells using the well established methods known in the art. In addition, water-soluble proteins usually do not require special environments/experimental settings for analysis, such as a cellular structure, e.g. cell membranes, whereas membrane proteins need to be in the context of cell membranes for their proper function and analysis. All of these combined make water-soluble proteins a more attractive target for research. For example, three-dimensional structures have been identified for more than 20,000 water-soluble proteins, whereas only around 20 membrane proteins have been identified at the three dimensional level. This clearly shows the difficulty associated with the study of membrane proteins.
The difficulty associated with the production, purification, and study of membrane proteins mainly stems from their structures and location in cells. The membrane proteins largely comprised of two parts; a hydrophilic part that is present outside of the lipid layer of a cell membrane and a hydrophobic part that is embedded within the lipid layer. Accordingly, in order for the functional study of membrane proteins to be possible, experimental settings that may accurately reconstitute cell membranes as found in vivo are required. The experimental settings would also be able to provide an environment where hydrophilic and hydrophobic regions coexist and thus allows a formation of the proper tertiary structure of membrane proteins.
However, it is very difficult to reproduce such an environment in vitro where membrane proteins would be able to adopt a proper tertiary and/or quaternary structure and interact with other proteins in the membrane as they would do in vivo. Previous efforts to provide such an environment include the use of a cell membrane prepared by isolating the membrane components of cells employing a variety of surfactants but they did not bring satisfactory results [C. Dietrich, et al., Lipid raft reconstituted in model membranes, Biophys. J. 80, 1417-1428 2001].
This is partly explained by a recently developed theory called “the lipid-raft model”, in which signaling membrane molecules are thought to be compartmentalized rather than continuously drifted in cell membranes as suggested by the previous, widely accepted theory called “the fluid mosaic model”. The lipid-raft model has given a new insight into how the lipids and membrane proteins in cell membranes are distributed and had a profound impact on the structure and functional studies of membrane proteins. According to the fluid mosaic model, membrane proteins are considered continuously drifted in cell membranes having a lipid bi-layer structure, resulting in a random and uniform distribution of the components within cell membranes. According to the lipid-raft model, however, which is being increasingly supported by accumulating data from many studies, membrane proteins show a localized distribution within cell membranes, being located on a defined area of the cell membranes called a lipid domain, such as a lipid raft, where they specifically interact with other molecules to exert functions [Simons, K and Toomre, D. Lipid rafts and signal transduction, Nat. Rev. Mol. Cell Biol. 1, 31-41 2000; Brown, D. A. and London, E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts, J. Biol. Chem. 275, 17221-17224 2000].
Accordingly, considering that membrane proteins play a crucial role in various aspect of cell function, it is clear that a lipid domain including a lipid raft as suggested by the lipid-raft model would also play an important role for cellular functions, for example, intercellular communication, signal transductions, polarity of cells, cell fusion, and transport across cell membranes such as ion transfer. In support of this, recently, lipid raft domains are found closely involved in the development of human diseases such as senile dementia caused by the accumulation of amyloid beta, and bovine spongiform encephalopathy caused by the accumulation of a prion protein [Joanna M. Cordy, Ishrut Hussain, et al., Exclusively targeting-secretase to lipid rafts by GPI-anchor addition up-regulates-site processing of the amyloid precursor protein, PNAS 100, 11735-11740 2003].
Therefore, there is an urgent need for the development of an in vitro cell membrane model system that would be able to reconstitute and regulate the lipid domains in vitro in a way similar to those found in vivo and thus provide an environment that allows membrane proteins to function in vitro as they do in vivo. This will greatly enhance our understanding of membrane proteins as well as their interactions with other proteins or components, leading to the development of new therapeutic agents. Such a system would also require a precise control of the growth, size, and spatial distribution of the lipid domain including a lipid raft, enabling a more systematic large-scale in vitro study of the lipid-raft model. However, no such systems are developed in the art.
The conventional methods to analyze lipid domains are limited and usually considered very destructive, and include treating cells with harsh reagents such as Trition X-100 and extracting whole cell membranes followed by isolation of the lipid domains, discarding the rest of the membrane components [Comparisons of detergent extraction and confocal microscopy, Biophys. J. 89, 1102-1108 2005]. Furthermore the conventional methods do not provide any control over the selectivity for lipid domains and the spatial distribution and size of lipid domains including lipid rafts in the context of cell membranes.